Isolation of Immunocomplexes from Zebrafish Brain

Zebrafish is an excellent model to study vertebrate neurobiology, but its synaptic components that mediate and regulate fast electrical synaptic transmission are largely unidentified. Here, we describe methods to solubilize and immunoprecipitate adult zebrafish brain homogenate under conditions to preserve electrical synapse protein complexes. The methods presented are well-suited to probe electrical synapse immunocomplexes, and potentially other brain-derived immunocomplexes, for candidate interactors from zebrafish brain.


A. Tissue preparation-Freeze and store brains
Note: Adult fish should be euthanized according to proper guidelines for the ethical care and use of animals in research associated with IACUC protocols of the user's institution. 1. Euthanize an adult fish and dissect the brain out as described in Gupta and Mullins (2010; see detailed video at: https://www.jove.com/t/1717). 2. Transfer each dissected brain to an Eppendorf tube, immediately close the cap tightly, and snap freeze tissue by immersing the tube in liquid nitrogen for at least one minute.

B. DAY ONE-Homogenize brains
Note: The following protocol describes conditions for two IPs. It is important to maintain all reagents on ice throughout the procedure to prevent protein degradation. 1. Freshly prepare 5 mL of homogenization buffer by adding one protease inhibitor mini tablet and 5 µL of 1 M DTT; keep on ice. Note: Homogenization buffer is prepared in bulk and is sufficient for 50 brains. Use within 60 min of preparation to ensure optimal protease inhibitor activity and DTT stability during tissue homogenization. 2. Freshly prepare 5 mL of solubilization buffer by adding 5 µL of 1 M DTT; keep on ice.
Note: Solubilization buffer is prepared in bulk and is sufficient for 50 brains. Use within 60 min of preparation to ensure DTT stability during tissue solubilization. 3. Put five frozen zebrafish brains into an ice-cold 1 mL dounce tissue grinder.
Note: The resulting quantity of total protein from each brain will vary depending on the age and size of the adult fish dissected. As a general guideline, using two brains per IP often results in 1-2 mg total protein/IP, though the total quantity required will vary according to protein expression levels and antibody efficiency. 4. Add 0.5 mL of prepared ice-cold homogenization buffer.
Note: Save unused homogenization buffer at 4 °C for use on DAY TWO, step C10 to mix the blank solution needed for the protein assay. 5. Homogenize brain tissue with 10-20 strokes using the loose pestle and then 10-20 strokes using the tight pestle. Note: Homogenizing in the absence of detergent prevents oxidation of proteins. Also, if needed, glass homogenizers and pestles can be decontaminated between samples by rinsing extensively with distilled water, then 70% ethanol, and then distilled water again before drying with air. 6. Transfer the homogenate (0.5 mL) to a 1.5 mL Eppendorf tube using a 1,000 µL pipette tip. 7. Add 0.5 mL of prepared solubilization buffer and gently invert to thoroughly mix the solutions.
Note: The prepared solubilization buffer contains 4% detergent, so that when mixed with an equal volume of brain homogenate, the final detergent concentration is 2%. Save unused solubilization buffer at 4 °C for use on DAY TWO, step C10 to mix the blank solution needed for the protein assay. 8. Solubilize the brain tissue by rocking the tube on a benchtop rocker at 24 rpm, overnight at 4 °C.

C. DAY TWO-Preclear and set up immunoprecipitations
1. Prepare pre-washed Pierce Protein A/G agarose by washing a 250 µL bed volume with 1 mL of preclear buffer. Note: Pre-washed Pierce Protein A/G agarose is prepared in bulk and is sufficient for three preparations. 2. Spin at 1,000 × g for 3 min at 4 °C. 3. Remove buffer and repeat wash steps C1 and C2 two more times using new preclear buffer each time. 4. After the final wash, remove buffer and resuspend agarose in 250 µL of preclear buffer to achieve a 500 µL 50% slurry of pre-washed Pierce Protein A/G agarose. Store pre-washed agarose at 4 °C until use in step C7 below and in DAY THREE, step D2. 5. Next, centrifuge the overnight solubilized brain homogenate at 20,000 × g for 30 min at 4 °C. 6. Without disturbing the pellet, transfer the solubilized brain supernatant to a new 1.5 mL Eppendorf tube using a 1,000 µL pipette tip. 7. Preclear the brain supernatant by adding 100 µL of 50% slurry of pre-washed Pierce Protein A/G agarose (prepared in steps C1-C4), and rock on benchtop rocker at 24 rpm for 1 h at 4 °C. 8. Spin the supernatant at 1,000 × g for 3 min at 4 °C to pellet the agarose.

D. DAY THREE-Capture immunoprecipitates
1. Centrifuge the IPs at 1,000 × g for 1 min at 4 °C. 2. Add 30 µL of 50% slurry of pre-washed Pierce Protein A/G agarose (prepared in DAY TWO steps C1-C4 and stored at 4 °C) to each IP. 3. Rock IPs on a benchtop rocker at 24 rpm for 1 h at 4 °C. 4. Centrifuge IPs at 1,000 × g for 3 min at 4 °C. 5. Remove supernatant with a 1,000 µL pipette tip without disturbing the agarose. 6. Add 1 mL of IP wash buffer to the agarose and invert several times. 7. Spin 1,000 × g for 3 min at 4 °C. 8. Repeat wash steps D5-D7 two more times, using new IP wash buffer each time. 9. On the last wash, completely remove the wash buffer from the agarose using a 200 µL pipette tip, followed by a flattened 10 µL gel loading tip. 10